PB2523: DI-2-ETHYLHEXYL PHTHALATE (DEHP) INHIBITS HEMOGLOBIN S POLYMERIZATION
Authors: Rodrigo Abreu Camacho, Aghata Vitoria Machado, Fernanda de Oliveira Mendonça, Lyzes Rosa Teixeira-Alves, Camila Cristina Guimarães-Nobre, Evelyn Mendonça Reis, Thyago Rubens Cardim Pires, Priscila Ferreira Da Silva, Leandro Miranda-Alves, Clemilson Berto-Junior
Journal: HemaSphere (2023)

Topic: 26. Sickle cell disease Background: Sickle cell disease (SCD) is a hereditary hemoglobinopathy, caused by a mutation at position 6 of the β-globin chain, where a glutamic acid is replaced by a valine, giving rise to hemoglobin S (HbS). When deoxygenated, HbS polymerizes by interaction of valine 6 with adjacent beta chain leading to formation of a sickle-shaped erythrocyte. To increase life expectancy, several treatments are used such as blood transfusion. Blood bags are made of polyvinyl chloride (PVC), and the addition of plasticizers is very common in order to ensure greater flexibility of the material and help in its preservation. The most widely used plasticizers are phthalates, manly di (2-ethylhexyl) phthalate (DEHP). DEHP is found in many products as gloves, dentistry products, toys, food containers and is a known endocrine disruptor with widespread effects in several tissues but it lacks effects on SCD erythrocytes as these patients are subject to numerous transfusions during their life. Aims: The present work aimed to determine whether DEHP affects HbS polymerization and the mechanism implicated in this possible effect Methods: The blood of patients with SCD was collected and the washed 3 times with PBS and buffy coat was discarded. Packed red blood cells (RBC) was used for polymerization assay by placing it in a 96-well plate along with DEHP in four concentrations (25 uM, 2.5 uM, 250 nM and 25 nM) and sodium metabisulfite. Readings were performed for 30 minutes every minute. We also performed polymerisation with purified HbS at the same DEHP concentrations and the same conditions. The docking of DEHP with the HbS was also performed with beta subunit (1DGF) using Chimera software for protein preparation, PyRx for docking, and BIOVIA Discovery Studio for visualisation. For circular dichroism, far UV-CD spectra (190-260 nm) was obtained at 37ºC and recorded in a 1-mm quartz cell with HbS at 1 mg/mL in phosphate buffer. Transmission electron microscopy was performed with samples adhered to a carbon-coated grid, blotted, and stained for 10 minutes with 2% uranyl acetate in water and protein at 1 mg/mL. Results: DEHP was able to reduce HbS polymerization in all concentrations used in whole erythrocyte assay. To assess whether this effect is mediated by direct interaction to HbS or other cell components, molecular docking simulations were performed and demonstrated that DEHP interacts with several amino acid residues and most importantly with valine 6 by carbon hydrogen bonding. When analyzing purified HbS polymerization to confirm last result, we observed that DEHP decreased polymer formation by means of interacting with HbS itself since this assay also demonstrated decreased polymerization. Circular dichroism showed HbS losing alpha helix and beta leaflets after deoxygenation that is partially restored in the presence of DEHP, and transmission electron microscopy highlighted that DEHP decreased polymer formation when compared to those in deoxygenated samples. Summary/Conclusion: In conclusion, we can infer which DEHP, that is present in transfusion blood bags, in low concentrations can decrease the formation of HbS polymers in vitro in erythrocytes from SCD patients, emphasizing the need to abroad the influence of endocrine disruptors’ influence specifically for the SCD population Keywords: Sickle cell disease